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The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFβ, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN overexpression in the decidua is associated with preeclampsia (PE); this may have a causal role in PE by compromising endovascular differentiation of the trophoblast and uterine angiogenesis, resulting in poor arterial remodeling. Elevated DCN level in the maternal blood is suggested as a potential biomarker in PE.

Introduction: Invasion of extravillous trophoblasts (EVT) is tightly linked to appropriate cell to cell contact as well as paracrine guidance of EVT by maternal uterine cells, conducted by a variety of locally expressed cytokines. Here we investigated the interaction of the first trimester trophoblast cell line ACH-3P with adult iliac arterial (AEC) and venous endothelial cells (VEC)., Methods: The impact of ACH-3P conditioned medium (Cdm), obtained at 2.5% and 21% oxygen, on endothelial cell viability (LDH-Assay) and network formation (Matrigel-Assay) was tested. We investigated cytokine expression of AEC- and VEC-Cdm and confirmed results with ELISA analysis, and investigated the influence of Cdm on ACH-3P proliferation and invasion. Additionally, direct co-culture experiments with ACH-3P and AEC on Matrigel were performed. A subset of experiments was verified with primary trophoblasts as well as with first trimester placenta in situ specimens., Results: ACH-3P-Cdm significantly enhanced cell viability of AEC and VEC after 72 h. ACH-3P-Cdm at 2.5% oxygen stabilized endothelial network structures in Matrigel up to 24 h, similar to the effect of a direct co-culture of AEC and ACH-3P. AEC and VEC showed a similar pattern of secreted cytokines. However, elevated levels of cytokines secreted by AEC were found for GRO, IL-6, MMP-1 and uPAR. ELISA confirmed elevated concentrations of IL-6 and uPAR in AEC compared to VEC. ACH-3P and primary trophoblasts more likely invaded towards AEC-Cdm than towards VEC-Cdm. Addition of IL-6 to Cdm increased the invasion potential of both cell types. AEC- and VEC-Cdm reduced ACH-3P cell proliferation after 24 h of culture. IL-6 was highly expressed in uterine AEC compared to VEC as visualized by immunohistochemistry., Discussion: The presented results clearly demonstrate that cytokines of both cell types, AEC and trophoblasts, differentially contribute to successful guidance and interaction in the process of trophoblast invasion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Abstract: Autophagy, a process for cellular cleaning through the removal of intracellular components in lysosomes, is a well conserved mechanism from yeast to mammalian cells, and also contributes to the maintenance of cellular homeostasis and of the energetic balance, in cellular and tissue remodeling, and cellular defense against extracellular insults and pathogens. The role of autophagy in placentation has been clarified. Autophagy is induced in trophoblasts under physiological hypoxia during early pregnancy and seems to have a role in placentation. Recent findings suggest that impaired autophagy might induce poor placentation in preeclamptic cases. In this review, we discuss the role of autophagy and summarize the role of autophagy-related genes in placentas. [Copyright &y& Elsevier]

Discarded at birth, the placenta is a highly complex and fascinating organ. During the course of a pregnancy, it acts as the lungs, gut, kidneys, and liver of the fetus. The placenta also has major endocrine actions that modulate maternal physiology and metabolism and provides a safe and protective milieu in which the fetus can develop. The human placenta undergoes dramatic transformations in form and function between the first trimester, when organogenesis occurs, and the remainder of pregnancy that reflect evolutionary responses to changing oxygen concentrations in the earth's atmosphere. Recent research indicates a more interactive dialogue between the placenta and the maternal tissues than previously recognized. The endometrial glands provide histotrophic support during the first weeks of pregnancy, and the placenta appears able to stimulate its own development by up-regulating gland activity in response to endocrine signals. Extravillous trophoblast cells migrate from the placenta into the uterine wall, in which they interact with cells of the maternal innate immune system. These interactions have a physiological, rather than a classical immunological, outcome and most probably mediate remodeling of the uterine spiral arteries that supply the placenta. Furthermore, deportation of aggregates of transcriptionally active trophoblast nuclei, and the release of exosomes carrying microribonucleic acids challenge our perceptions of fetal-maternal signaling and where the placental interface actually lies. Here we reconsider definitions of the placenta in the light of these recent advances., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: For ectopic tubal pregnancy to be viable, it requires a supporting vascular network and functioning trophoblast. Slit2/Robo1 signaling plays an important role in placental angiogenesis during normal pregnancy. Hence, we here investigated whether or not Slit2/Robo1 signaling also had an impact in ectopic tubal pregnancy., Methods: The Slit2 and Robo1 expression pattern relevant to trophoblast invasive behavior and vascular remodeling was studied in human tubal placenta obtained from patients with ectopic pregnancy (5-8weeks gestation), The trophoblast development, vascular architecture and Robo1 expression pattern were observed in Slit2 overexpression (Slit2-Tg) and C57BL mice placenta (E13.5 and E15.5)., Results: Marked with CK-7 and Vimentin, the vessel profiles of fallopian tube were classified into four stages. In the presence of extravillous trophoblast (EVT), stellate-shaped and polygonal-shaped EVTs were observed, and the stellate-shaped EVT showed the higher Slit2 expression (P < 0.01) but lower Robo1 expression (P < 0.05) than polygonal-shaped cells. By contrast, a temporary Slit2 up-regulation in remodeling vessel and Slit2 down-regulation in remodeled vessel of polygonal-shape extravillous trophoblast cells occurred in tubal pregnancies. In Slit2-Tg mice E13.5 and E15.5 placenta, Slit2 overexpression promoted vascular remodeling by increasing in the diameter of the maternal blood sinusoids and fetal capillaries, but enhanced the thickness of trophoblast and vasculature at E15.5 Slit2-Tg mice., Conclusions: The varying Slit2 and Robo1 expression in EVTs was associated with trophoblast invasion and probably plays an important role in the events of blood vessel remodeling of the fallopian tube tissues., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown., Methods: IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used., Results: We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR(1), induced IL-8 gene expression but not protein secretion. The specific S1PR(2) inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR(1)/S1PR(3) antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells., Discussion: In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR(1) and its secretion is regulated through S1PR(2) receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Study Question: Does inhibition of dimethylarginine dimethylaminohydrolase (DDAH) increase the sensitivity of trophoblasts to TRAIL-induced apoptosis?, Summary Answer: Inhibition of DDAH1, but not DDAH2, increases the sensitivity of trophoblasts to TRAIL-induced apoptosis., What Is Known Already: Successful human pregnancy is dependent on adequate trophoblast invasion and remodelling of the maternal spiral arteries. Increased trophoblast apoptosis is seen in pregnancies complicated by pre-eclampsia. The mechanism underlying this increase is unknown. We have previously shown that nitric oxide (NO) is involved in regulating trophoblast motility and invasion, and have also demonstrated an important role for NO in regulating trophoblast sensitivity to apoptotic stimuli. DDAH is an enzyme that metabolizes asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, previously shown to be elevated in the plasma of pre-eclamptic mothers., Study Design, Size, Duration: This study used the human extravillous trophoblast-derived cell line SGHPL-4 cells. All experiments were performed at least three times., Participants/materials, Setting, Methods: The effect of DDAH on trophoblast apoptosis was examined using siRNA and time-lapse microscopy. Changes in the expression of DDAH were followed by PCR and western blot analysis. Receptor expression was followed by flow cytometry., Main Results and the Role of Chance: Inhibiting the expression of DDAH1, but not DDAH2, resulted in a significant increase in the sensitivity of the EVT cell line SGHPL-4 to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis (P < 0.01). This response could be mimicked by the addition of Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis and the substrate for both isoforms of DDAH. We further showed that this increased sensitivity to apoptosis is accompanied by a significant increase in the expression of TRAIL receptor 2 (TR2; P < 0.05) but not TRAIL receptor 1 (TR1)., Limitations, Reasons for Caution: This study was performed only in vitro using a well characterized trophoblast cell line, SGHPL-4, derived from first trimester extravillous trophoblasts., Wider Implications of the Findings: This study provides new insight into the role of the DDAH/ADMA pathway in the regulation of trophoblast function. Both dysregulation of DDAH and the accumulation of ADMA have been associated with the development of pre-eclampsia. This is the first study to implicate the DDAH/ADMA pathway as a mechanism that might underlie the poor trophoblast invasion seen in this common pregnancy disorder., Study Funding/competing Interests: B.A.L. was supported by a grant from Action Medical Research UK (SP4577). A.E.W. was supported by a grant from the Wellcome Trust (091550). There are no competing interests and the authors have no conflict interest to declare., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

The process of uterine spiral artery remodeling in the first trimester of human pregnancy is an essential part of establishing adequate blood perfusion of the placenta that will allow optimal nutrient/waste exchange to meet fetal demands during later development. Key regulators of spiral artery remodeling are the uterine natural killer cells and the invasive extravillous trophoblasts. The functions of these cells as well as regulation of their activation states and temporal regulation of their localization within the uterine tissue are beginning to be known. In this review, we discuss the roles of these two cell lineages in arterial remodeling events, their interaction/influence on one another and the outcomes of altered temporal, and spatial regulation of these cells in pregnancy complications., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Notch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations., Methods: Localisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)(+) and differentiated HLA-G(+) CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage., Results: In tissue sections Notch2 expression was higher in HLA-G(+) distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G(+) CTBs compared to EGFR(+) CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility., Discussion: Notch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell-cell contact of neighbouring dCCTs, could attenuate trophoblast migration., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Study Question: Do trophoblast subtypes differ in their expression of erythroblastic leukaemia viral oncogene homologue (ERBB) receptor family members and responsiveness towards specific growth factor ligands?, Summary Answer: Our data reveal a reciprocal expression pattern of epidermal growth factor receptor (EGFR)/ERBB4 in proliferative and ERBB2/ERBB3 in invasive trophoblast subtypes, as well as a restricted responsiveness to epidermal growth factor (EGF) and heparin-binding (HB)-EGF., What Is Known Already: EGFR is expressed by villous cytotrophoblasts (vCTBs), but absent from extravillous trophoblasts (EVTs), which specifically up-regulate ERBB2., Study Design, Size, Duration: Tissue samples of human first trimester placentae (n = 50) and deciduae (n = 5) obtained from elective pregnancy terminations were used to study trophoblast subtype-specific ERBB receptor expression and responsiveness to recombinant human EGF and HB-EGF. Age-matched complete hydatidiform mole (CHM) placentae (n = 12) were assessed for EGFR and ERBB4 expression in proliferation-competent regions., Participants/materials, Setting, Methods: ERBB receptor expression was analysed in primary trophoblast cell isolates by means of microarray, quantitative real-time PCR and western blotting, as well as immunofluorescence stainings of placental and decidual tissue sections. EGF and HB-EGF were tested for their potential to activate ERBB receptors in purified EGFR(+) and HLA-G(+) trophoblasts. 5-Ethynyl-2'-deoxyuridine incorporation assays were performed to study the effect of both ligands on the proliferative capacity of primary trophoblasts as well as of vCTBs and proximal cell column trophoblasts (pCCTs) in placental floating explants. Finally, the average number of EGFR(+) vCTB and pCCT layers was determined in CHM placentae and compared with healthy age-matched controls., Main Results and the Role of Chance: Proliferative vCTBs and pCCTs co-express EGFR and ERBB4, but are devoid of ERBB2 and ERBB3. In contrast, HLA-G(+) trophoblast subtypes exhibit an EGFR/ERBB4(-) and ERBB2/ERBB3(+) phenotype. EGF and HB-EGF activate EGFR, ERBB4, AKT and extracellular signal-regulated kinase 1/2 in EGFR(+) primary trophoblasts; however, they do not show an effect on HLA-G(+) EVTs. Both ligands strongly induce cell cycle progression in primary trophoblasts (P < 0.05) and placental explant-associated vCTBs (P < 0.05) and pCCTs (P < 0.05). Notably, EGFR(+) vCTB (P < 0.0001) and pCCT (P < 0.0001) layers are significantly expanded in CHM placentae when compared with healthy controls., Limitations, Reasons for Caution: Cells were removed from their physiological context and may therefore respond differently to various stimuli., Wider Implications of the Findings: In this study we define EGFR as a marker for proliferative trophoblast subtypes within the human placenta. Manipulation of EGFR signalling might thus offer a promising therapeutic avenue for the treatment of molar pregnancies associated with trophoblast hyperplasia., Study Funding/competing Interests: This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P.). There are no competing interests to declare., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation., Methods: This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), and its precursor, 25-hydroxyvitamin D3 (25-D3), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0-10 nM) or 25-D3 (0-100 nM) for 48-60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel(®) were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT., Results: EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged., Discussion: These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Abstract Objective: The maternal-fetal interface must modulate immune function to allow tolerance of fetal cells while still reacting to pathogens to suppress infection. Human leukocyte antigen-G (HLA-G) is a class Ib major histocompatibility complex protein involved in maternal-fetal tolerance. We posited that alterations in placental HLA-G expression predispose women to preterm birth. The aim of this study was to compare HLA-G expression in the maternal-fetal interface of term versus preterm human placentas., Methods: We performed a cross-sectional study of specimens from the basal plate of the human placenta from women enrolled in a tissue specimen and clinical data consortium. Immunohistochemistry with digital microscopic analysis was used to quantify HLA-G protein expression in the basal plate from preterm and term placentas., Results: Preterm birth <37 weeks occurred in 29.5% of 149 singleton pregnancies. HLA-G-positive cells occupied one-third of the basal plates, and the HLA-G-positive area was increased by 14% in placentas from preterm births than in those from term births (32.1% in term placentas versus 36.6% in preterm placentas)., Conclusion: Although HLA-G is required for maternal tolerance of the semi-allogeneic fetus, higher levels of HLA-G expression at the maternal-fetal interface is associated with preterm birth.

Objective: The purpose of this study was to determine whether fetuin-A affects trophoblast viability and invasion, whether growth factors that bind receptors that activate tyrosine kinase are impaired by fetuin-A, and whether elevated maternal serum fetuin-A levels are associated with adverse pregnancy outcomes. Study Design: We studied viability and invasion in first-trimester extravillous trophoblast cells that were exposed to fetuin-A, insulin-like growth factor, and placental growth factor. Insulin receptor substrates expression was assessed. We compared serum fetuin-A levels in 111 preeclampsia cases and 95 controls. Results: Fetuin-A reduced extravillous trophoblast cell viability and invasion in the presence or absence of growth factors. Fetuin-A reduced insulin receptor substrate–1 and tyrosine phosphorylated insulin receptor substrate–1 expression in extravillous trophoblast cells that had been treated with insulin-like growth factor. Elevated serum fetuin-A levels were more prevalent in preeclampsia cases than control subjects, even after we controlled for diabetes mellitus, hypertension, and obesity. Conclusion: Fetuin-A may decrease trophoblast viability and invasion caused by the inhibition of receptor tyrosine kinase activity. Elevated serum levels of fetuin-A may be associated with preeclampsia. [ABSTRACT FROM AUTHOR]

Objective: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. Methods: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFß1). The responsive gene to TGFß1 treatment was confirmed by quantitative real-time PCR. Results: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFß1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. Conclusion: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies. [Copyright &y& Elsevier]

Elastin breakdown in the walls of uterine spiral arteries during early pregnancy facilitates their transformation into dilated, high-flow, low-resistance channels. Elastin-derived peptides (EDP) can influence cell migration, invasion and protease activity, and so we hypothesized that EDP released during elastolysis promote extravillous trophoblast (EVT) invasion and further elastin breakdown. Treatment of the trophoblast cell line SGHPL4 with the elastin-derived matrikine VGVAPG (1 μg/ml) significantly increased total elastase activity, promoted migration in a wound healing assay and increased invasion through Matrigel-coated transwells compared with vehicle control (0.1% DMSO) or the scrambled sequence VVGPGA. Furthermore, treatment of first-trimester placental villous explants with this EDP significantly increased both the area of trophoblast outgrowth and distance of migration away from the villous tips. Primary first-trimester cytotrophoblast exposed to VGVAPG (1 μg/ml) for 30 min showed increased phosphorylation of endothelial nitric oxide synthase and activation of the mitogen activated protein kinase pathway, events also associated with tumour cell migration and invasion. These in vitro observations suggest liberation of bioactive EDP during induction of elastolysis in the uterine spiral arteries may orchestrate a positive feedback loop that promotes EVT invasion and further elastin breakdown, contributing to the process of vascular remodelling., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy?, Summary Answer: T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy., What Is Known Already: Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface., Study Design, Size, Duration: This laboratory-based study used human decidua from first (8-11 weeks; n = 18) and second (12-16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies., Participants/materials, Setting, Methods: Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10-) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel(®) was evaluated., Main Results and the Role of Chance: Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro., Limitations, Reasons for Caution: Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo., Wider Implications of the Findings: Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory 'switch' in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes., Study Funding/competing Interest(s): This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research - Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.

Shallow trophoblast invasion and impaired vascular remodeling of spiral arteries have been recognized in early-onset preeclampsia. Placentation and vascular remodeling are multistep processes, and hypoxia, placental oxidative stress, excessive or atypical maternal immune response to trophoblasts, exaggerated inflammation, and increased production of anti-angiogenic factors such as the soluble form of the vascular endothelial growth factor (VEGF) receptor (sFlt-1) and soluble endoglin (sENG) may play a role in poor placentation in preeclampsia. Recent findings suggest that autophagy plays an important role in extravillous trophoblast (EVT) invasion and vascular remodeling under hypoxia, and sENG inhibits EVT invasion and vascular remodeling by the inhibition of autophagy under hypoxic conditions. In this review, we discuss the relationship between inadequate autophagy and poor placentation in preeclampsia., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Nodal, a secreted signaling protein from the transforming growth factor beta (TGF-β)-super family plays a vital role during early embryonic development. Recently, it was found that maternal decidua-specific Nodal knockout mice show intrauterine growth restriction (IUGR) and preterm birth. The chromosomal location of NODAL is in the same linkage area as the placental (fetal) pre-eclampsia (PE) susceptibility gene STOX1, which is associated with the familial form of early-onset, IUGR-complicated PE. As the STOX1 linkage was originally identified in women being born from a pre-eclamptic pregnancy as well as suffering from PE themselves, the linkage could in part be caused by NODAL, which is why the potential maternal-fetal interaction between STOX1 and NODAL was investigated. In the PE families with the STOX1 susceptibility allele carried by the children born from pre-eclamptic pregnancies, it was found that the pre-eclamptic mothers themselves all carried the NODAL H165R SNP, which causes a 50% reduced activity. Surprisingly, in decidua-specific Nodal knockout mice the fetal placenta showed up-regulation of STOX1 and NODAL expression. Conditioned media of human first trimester decidua and a human endometrial stromal cell line (T-HESC) treated with siRNAs against NODAL or carrying the H165R SNP were also able to induce NODAL and STOX1 expression when added to SGHPL-5 first trimester extravillous trophoblast cells. Finally, a human TGF-β/BMP signaling pathway PCR-array on decidua and the T-HESC cell line with Nodal knockdown revealed upregulation of Activin-A, which was confirmed in conditioned media by ELISA. We show that maternal decidua Nodal knockdown gives upregulation of NODAL and STOX1 mRNA expression in fetal extravillous trophoblast cells, potentially via upregulation of Activin-A in the maternal decidua. As both Activin-A and Nodal have been implicated in PE, being increased in serum of pre-eclamptic women and upregulated in pre-eclamptic placentas respectively, this interaction at the maternal-fetal interface might play a substantial role in the development of PE.

Study Question: What are the effects of the eotaxin group of chemokines (CCL11, CCL24 and CCL26) on extravillous trophoblast (EVT) functions important during uterine decidual vessel remodelling?, Summary Answer: CCL11, CCL24 and CCL26 can regulate EVT migration, invasion and adhesion, highlighting a potential regulatory role for these chemokines during uterine decidual spiral arteriole remodelling in the first trimester of human pregnancy., What Is Known Already: A successful human pregnancy depends on adequate remodelling of the uterine decidual spiral arterioles, a process carried out by EVT which invade from the placenta. The invasion by EVT into the maternal uterine decidual vessels is regulated by the interaction of many factors including members of the chemokine subfamily of cytokines., Study Design, Size, Duration: This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions., Participants/materials, Setting, Methods: The effect of recombinant human CCL11, CCL24 and CCL26 on EVT migration and invasive potential was measured using the xCELLigence real-time system, wound-healing and Matrigel invasion assays, zymography to measure MMP activity and reverse zymography to measure TIMP activity. A commercially available adhesion assay was used to assess EVT adhesion to extracellular matrix proteins., Main Results and the Role of Chance: All the three eotaxins were found to significantly stimulate migration of the EVT-derived cell line HTR8/SVneo (P < 0.05) with no significant changes in cell number following treatment with each chemokine (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo invasion (P < 0.05) and MMP2 activity (P < 0.05) without any effects on TIMP2 activity (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo cell binding to collagen IV (P < 0.05) and fibronectin (P < 0.05)., Limitations, Reasons for Caution: This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo., Wider Implications of the Findings: This study is the first to comprehensively examine the effects of the eotaxin group of chemokines on EVT functions and demonstrates that all the three eotaxins have the ability to regulate EVT functions critical to their role in vessel remodelling. This identifies a new role for the eotaxin group of chemokines during placentation.

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.

Remodeling of maternal spiral arteries by invasion of extravillous trophoblast (EVT) is crucial for an adequate blood supply to the fetus. EVT cells that migrate through the decidual tissue destroy the arterial muscular lining from the outside (interstitial invasion), and those that migrate along the arterial lumen displace the endothelium from the inside (endovascular invasion). Numerous factors including cytokines/growth factors, chemokines, cell adhesion molecules, extracellular matrix-degrading enzymes, and environmental oxygen have been proposed to stimulate or inhibit the differentiation/invasion of EVT. Nevertheless, it is still difficult to depict overall pictures of the mechanism controlling perivascular and endovascular invasion. Potential factors that direct interstitial trophoblast towards maternal spiral artery are relatively high oxygen tension in the spiral artery, maternal platelets, vascular smooth muscle cells, and Eph/ephrin system. On the other hand, very little is understood about endovascular invasion except for the involvement of endothelial apoptosis in this process. Only small numbers of molecules such as polysialylated neural cell adhesion molecules and CCR1 have been suggested as specific markers for the endovascular trophoblast. Therefore, an initial step to approach the mechanisms for endovascular invasion could be more detailed molecular characterization of the endovascular trophoblast.

Human extravillous trophoblasts (EVT) invade maternal deciduas and reconstructed maternal spiral arteries during early placentation. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVT from further invasion have not been well understood. Recently, it was found that EVT that had already ceased their invasion, specifically expressed cluster of differentiation (CD9) and dipeptidyl peptidase IV (DPPIV) on their cell surface. In addition, EVT migrating to maternal spiral arteries expressed CC chemokine receptor type-1 (CCR-1), which is a chemokine receptor for regulated on activation normal T cell expressed and secreted (RANTES) and so on. CD9 is associated with integrin molecules on the cell surface and is considered to modulate integrin function. In contrast, DPPIV is a cell surface peptidase that can metabolize RANTES at extracellular sites before its accessing to the chemokine receptors. In vitro functional assay showed that CD9, DPPIV and RANTES are involved in the regulation for EVT invasion. From these findings, it can be proposed that CD9 and DPPIV, including chemokines, are new regulatory factors for human extravillous trophoblasts. (Reprod Med Biol 2005; 4 : 189-195).